1. Field of the Invention
The present invention relates to a process for producing peroxidases from the fungus Phanerochaete chrysosporium.
2. Description of the Background
Phanerochaete chrysosporium is a fungus associated with "white rot of wood". It is a hymenomycete which belongs to the order Aphylophorales and to the family Corticaceae. It has the property of degrading lignin to the point of mineralization (final products: CO.sub.2 +H.sub.2 O).
This fungus produces exocellular peroxidases: the latter comprise, in particular, isozymes of manganese peroxidase (MnPs) [KUWAHARA et al. FEBS Let., 169, pp. 247-250, (1984)] and those of lignin peroxidase (LiPs) [TIEN M. and KIRK T. K., Science, 221, pp. 661-663, (1983); GLENN et al. Biochem. Biophys. Res. Commun., 114, pp. 1077-1083]. These enzymes are glycosylated haemoproteins whose average molecular mass is 40 kDa [LEISOLA et al., J. Biol. Chem., 262 pp. 419-424, (1984)].
Manganese peroxidases and lignin peroxidases are capable of catalysing the oxidation of numerous aromatic substrates, especially lignin, using hydrogen peroxide as cosubstrate. These properties find their main applications in the field of papermaking and that of waste treatment.
Hitherto, the lignin peroxidases are the ones which have chiefly been used in this type of application.
For example, French Patent 2,574,427 describes two strains of Phanerochaete chrysosporium possessing especially high lignin peroxidase activity, and their culture on a medium containing an assimilable nitrogen source as well as an assimilable carbon source and a source of assimilable inorganic salts.
The proposal has also been made (French Patent 2,600,077) to culture Phanerochaete chrysosporium on a basal culture medium supplemented with unsaturated fatty acids and/or natural amino acids. Other teams have proposed adding detergent of the Tween type [FAISON and KIRK, Appl. Environ. Microbiol. (1985), 49, pp. 299-304], or alternatively adding veratryl alcohol [LEISOLA et al., J. Biotechnol. (1985), 3, pp. 97-107], in order to increase the synthesis of lignin peroxidase.
The inventors' team has also, in previous investigations, demonstrated various parameters whose optimization enabled the production of lignin peroxidase to be increased: addition of oleic acid and/or of exogenous phospholipids, control of the culture temperature, and the like. These investigations led to the development of a process for producing lignin peroxidase, which forms the subject of European Patent 0,437,500.
This process comprises several successive steps, each of them being performed under different culture conditions; the first step is performed on synthetic medium comprising inorganic salts, a carbon source and a nitrogen source, in the presence of yeast extract, a source of phospholipids and emulsified fatty acids; the mycelium formed is then cultured in a partially renewed culture medium, supplemented with veratryl alcohol but not comprising emulsified fatty acids, and whose phospholipid content represents only 1/7 to 1/8 of that of the culture medium used in the first step; in a third step, the medium is replaced completely by a new medium, comprising the same proportion of phospholipids and of veratryl alcohol as that of the second step, and the carbon source, the nitrogen source and the yeast extract at 1/4 of their content in the medium of the first step; lastly, a fourth culture step is performed in a medium lacking yeast extract, carbon source and emulsified fatty acids, but comprising the same proportion of phospholipids and of veratryl alcohol as that of the preceding steps.
The lignin peroxidase may then be recovered from the culture medium. The use of this process enables the enzyme content to be increased very significantly; the lignin peroxidase activity in the medium is approximately 240 U per liter and per day.
The research performed hitherto has mainly been concerned with increasing the lignin peroxidase activity. However, the manganese peroxidases are being seen increasingly to be capable of performing a key role in the bioconversion of polymeric aromatic compounds of the lignin type. In effect, contrary to lignin peroxidases whose activity is limited by the low penetration of the lignified walls by the enzyme, manganese peroxidases act via species of low molecular weight which diffuse readily. Briefly, the catalytic cycle of manganese peroxidases involves the oxidation of Mn(II) to Mn(III) which, after complexing with organic acids, generates diffusing oxidizing species capable of depolymerizing natural lignin [WARIISHI K., et al., Biochem. Biophys. Res. Comm. 176, pp. 269-276, (1991)].
BONNARME and JEFFRIES [J. Ferment. Bioeng. 70:158-163 (1990)] studied the regulation of the production of Phanerochaete chrysosporium lignin peroxidase and manganese peroxidase under different culture conditions; they observed in this way that the amount of these enzymes varied especially in accordance with the Mn(II) concentration: at low concentration, lignin peroxidase is produced preferentially (760 nmol/ml.min under optimal conditions), while at high concentration, the production of manganese peroxidase (950 nmol/ml.min under optimal conditions) is the one which is favoured.